The fundamental objectives of the proposed research are: (1) to further broaden our understanding of the detailed molecular structure and mechanism of action of the enzyme dihydrofolate reductase (DHFR), and (2) to begin applying this knowledge toward the design of specific chemotherapeutic agents. We propose to extend our X-ray diffraction studies of bacterial DHFRs to include DHFR from chicken liver and a nonchromosomal DHFR specified in E. coli by R plasmid R67. The chicken liver enzyme can be crystallized in the presence (or absence) of a variety of substrates, cofactors and inhibitors thus providing an opportunity to study how these small molecules interact with the enzyme in the different complexes. It should then be possible to rationalize from a structural standpoint the well-documented diverse binding affinities exhibited for various small molecule inhibitors by bacterial DHFRs on the one hand and vertebrate DHFRs on the other. New inhibitors will be designed and synthesized in a rational effort to develop a clinically useful antibiotic against Neisseria gonorrhoeae.